Describe How Quorum Sensing Can Lead to Formation of a Biofilm.

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Peer-reviewed

Inquiry Article

The Evolution of Quorum Sensing in Bacterial Biofilms

• Carey D Nadell,
• Joao B Xavier,
• Simon A Levin,
• Kevin R Foster

x

• Published: January 29, 2008
• https://doi.org/10.1371/periodical.pbio.0060014

Figures

Abstruse

Bacteria have fascinating and diverse social lives. They display coordinated grouping behaviors regulated past quorum-sensing systems that detect the density of other bacteria effectually them. A key example of such grouping behavior is biofilm formation, in which communities of cells attach to a surface and envelope themselves in secreted polymers. Curiously, later on reaching loftier cell density, some bacterial species activate polymer secretion, whereas others terminate polymer secretion. Here, we investigate this hit variation in the first evolutionary model of quorum sensing in biofilms. Nosotros utilize detailed individual-based simulations to investigate evolutionary competitions between strains that differ in their polymer production and quorum-sensing phenotypes. The benefit of activating polymer secretion at high cell density is relatively straightforward: secretion starts upon biofilm germination, allowing strains to push their lineages into food-rich areas and suffocate neighboring cells. Simply why use quorum sensing to finish polymer secretion at high jail cell density? We find that deactivating polymer production in biofilms can yield an advantage by redirecting resources into growth, but that this reward occurs only in a limited time window. We predict, therefore, that down-regulation of polymer secretion at loftier cell density volition evolve when it can coincide with dispersal events, only it will exist disfavored in long-lived (chronic) biofilms with sustained competition among strains. Our model suggests that the observed variation in quorum-sensing beliefs tin can be linked to the differing requirements of bacteria in chronic versus acute biofilm infections. This is well illustrated by the case of Vibrio cholerae, which competes within biofilms by polymer secretion, terminates polymer secretion at high cell density, and induces an acute illness course that ends with mass dispersal from the host. More generally, this work shows that the residuum of competition inside and among biofilms tin be pivotal in the evolution of quorum sensing.

Writer Summary

Bacteria are increasingly recognized equally highly interactive organisms with complex social lives, which are critical to their capacity to crusade disease. In item, many species inhabit dumbo, surface-leap communities, termed biofilms, inside which they communicate and respond to local cell density through a procedure known every bit quorum sensing. Enormous endeavor has been devoted to understanding the genetics and biochemistry of biofilm germination and quorum sensing, but how and why they evolve remain about unexplored. Many bacteria apply quorum sensing to regulate the secretion of mucilaginous extracellular slime, an integral feature of biofilm life. Intriguingly, notwithstanding, some pathogenic species plow on slime production at high prison cell density, whereas others turn information technology off. Using an individual-based model of biofilm growth, we investigated why dissimilar species apply quorum sensing to control slime product in opposite ways. The undercover underlying this variation appears to reside in the nature of infections. Turning slime on at high prison cell density can permit one strain to suffocate another when contest is intense, as occurs in long-lived chronic infections. Meanwhile, turning slime secretion off at loftier cell density tin can do good a strain causing an acute infection by allowing rapid growth before departing the host.

Commendation: Nadell CD, Xavier JB, Levin SA, Foster KR (2008) The Evolution of Quorum Sensing in Bacterial Biofilms. PLoS Biol 6(1): e14. https://doi.org/10.1371/journal.pbio.0060014

Academic Editor: Nancy A. Moran, University of Arizona, United States of America

Received: Oct 2, 2007; Accepted: December 10, 2007; Published: January 29, 2008

Copyright: © 2008 Nadell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original writer and source are credited.

Funding: CDN is supported by a Centennial Fellowship and Robert May Fellowship from Princeton University. KRF and JX are supported by National Institute of Full general Medical Sciences Center of Excellence Grant 5P50 GM 068763–01. SAL is pleased to acknowledge support from the Defense Advanced Research Projects Agency (DARPA) under grant HR0011-05-1–0057.

Competing interests: The authors take declared that no competing interests exist.

Abbreviations: EPS, extracellular polymeric substance; EPS⁺, constitutive extracellular polymeric substance-producing strain; EPS⁻, not-extracellular polymeric substance-producing strain; QS⁺, quorum-sensing strain

Introduction

Once perceived as organisms that rarely interact, bacteria are at present known to lead highly social lives [1–iii]. Central to this sociality is an power to detect local cell density and thereby coordinate grouping behaviors [4–6]. This ability, termed quorum sensing, functions through the secretion and detection of autoinducer molecules, which accrue in a cell density-dependent manner. When autoinducer concentrations reach a threshold level, quorum-sensing cells answer, allowing them to modulate behaviors whose efficacy and fitness benefits depend upon the presence, or absenteeism, of other cells. Traits under quorum-sensing control include surface attachment [7], extracellular polymer product [8–10], biosurfactant synthesis [11], sporulation [12], competence [xiii], bioluminescence [14,15], and the secretion of nutrient-sequestering compounds and virulence factors [sixteen–18]. Quorum sensing is besides phylogenetically widespread, which suggests an early on origin in bacterial development [19].

In addition to sensing and responding to the presence of other cells, many bacteria class multicellular surface-bound aggregates, or biofilms, whose remarkable feats of persistence are the scourge of both medicine and manufacture [5,6,20–24]. Accordingly, biofilms confer on their members considerable advantages, including the ability to resist challenges from predators, antibiotics, and host allowed systems [half dozen,20,25–27]. Quorum sensing and biofilm germination are often closely linked, and it is likely that their interaction is central to the pathogenesis of many bacterial infections [eight–10,28–30]. The effects of quorum sensing, however, are highly variable and depend upon both the species under observation and the experimental conditions [28]. Four studies have emphasized how the potential for contest and conflict among strains of leaner tin shape the development of quorum sensing [31–34], only none take addressed biofilm germination. An open up challenge for microbiology, therefore, is to disentangle the ecological and evolutionary processes that drive quorum sensing and biofilm phenotypes and, in detail, their interaction.

A defining feature of many biofilm-forming bacteria is the secretion of extracellular polymeric substances (EPS). These polymers, which consist largely of polysaccharide and smaller amounts of protein and DNA, grade a matrix in which bacterial cells are embedded [5,6]. Recent empirical and theoretical work has shown that by secreting EPS, individual bacteria tin can both help and harm cells in their neighborhood and strongly touch the evolutionary dynamics within biofilms [35–38]. Using an private-based biofilm simulation framework, Xavier and Foster [36] demonstrated that EPS production can provide an advantage to secreting strains by allowing them to push their descendent cells upwardly into areas of loftier food availability while suffocating whatever neighboring cells that do not produce EPS.

EPS secretion is under quorum-sensing command in a number of bacterial model systems. Many species, including the pathogen Pseudomonas aeruginosa, activate EPS production at high cell density [8,10]. The evolutionary rationale for this strategy seems articulate: it increases the likelihood that polymer secretion will only occur in the biofilm state, where it affords a competitive reward, and not in the planktonic state, where it is presumably a waste of resources [36]. Unexpectedly, other species behave quite differently. The man enteric pathogen Vibrio cholerae initiates EPS secretion after attaching to a surface and losing flagellar activity [39,40]. Subsequently, in a style contrary to P. aeruginosa, 5. cholerae halts EPS secretion one time it reaches its high cell density quorum-sensing threshold [9,39]. Here, nosotros explore evolutionary explanations for this variability in quorum-sensing control of EPS production using an private-based model of biofilm formation [36]. In particular, nosotros ask why do some species actuate the biofilm-specific trait of polymer secretion at high cell density, while others terminate polymer secretion at high cell density?

Methods/Results

We follow pairwise evolutionary competitions between strains that differ both in their ability to produce extracellular polymeric substances (EPS) and the extent to which this behavior is under quorum-sensing control. For our simulation written report, we focus on iii strains with the post-obit behavior: (1) no polymer secretion and no quorum sensing (EPS⁻), (2) constitutive polymer secretion and no quorum sensing (EPS⁺), and (3) polymer secretion under negative quorum-sensing control such that EPS secretion stops at high cell density (QS⁺). A fourth strain for which polymer secretion is under positive quorum-sensing control is omitted from the main analysis because its behavior was institute to exist qualitatively identical to that of the EPS⁺ strain (see Word, Text S1, and Figure S1). Our simulations examine quorum-sensing control of a unmarried trait (EPS) in response to the concentration of a single autoinducer. In reality, bacteria often use more than 1 autoinducer to regulate multiple traits, and indeed, several quorum-sensing circuits may be linked via parallel or serial signaling pathways within the prison cell [15,16,41]. There is a rich telescopic, therefore, for additional study of many potential complexities of quorum-sensing–regulated social behaviors, which nosotros go out open up here.

Model Framework

Biofilm development involves a number of interacting physical and biological processes, including growth, neighbor-pushing, solute diffusion, and other prison cell–cell and jail cell–solute interactions, all of which occur largely at the spatial scale of single cells. Nosotros apply individual-based modeling methods to explore the emergent characteristics of these processes at the level of whole biofilms [42]. Simulated cells acquit independently co-ordinate to user-defined kinetic charge per unit expressions designed to represent the essential features of bacterial metabolism. Our simulations begin with one or more colonizing cells, which are fastened to a uniformly apartment surface and grow in a two-dimensional (two-D) infinite with horizontal periodic boundary conditions. The model framework used hither allows the definition of whatsoever number and kind of bacterial and solute species [43]. As cells consume substrate according to their strain-specific metabolism kinetics and produce boosted biomass, they grow and carve up one time a maximum cell radius is accomplished. Movement of cells, which are modeled as rigid circles, results from forces exerted between neighbors as they grow and divide. Summed over all the cells present, these forces cause the biofilm front to advance. Solutes diffuse across a boundary layer between the biofilm and a bulk fluid in which solute concentrations are assumed to be homogeneous and abiding. Inside this purlieus layer, we determine the dynamics of solute spatial distributions by solving the 2-D improvidence-reaction equations. In and then doing, we assume that solute concentrations attain their improvidence-reaction equilibria much faster than bacterial cells grow and split [43,44]. The biofilm simulation framework and its associated numerical methods accept previously been described in particular [42,43,45].

Strain Definitions

Following Xavier and Foster [36], we presume that leaner consume a substrate, S, and invest it in the production of biomass and EPS (for a full listing of model note, run across Table i). This allows a simple definition of the strains based upon their biomass versus EPS investment strategies. Non-EPS producers (EPS⁻) devote all substrate taken upwards to biomass product, whereas unconditional EPS producers (EPS⁺) ever allocate a proportion f to EPS synthesis.

Our third strain, QS⁺, is intended to represent a hypothetical kickoff step in the development of quorum sensing. We assume that QS⁺ cells have gained the ability to find a waste chemic produced past conspecific leaner. This chemical tin be envisioned as a byproduct of metabolism or cellular housekeeping that has been co-opted as a primitive autoinducer for monitoring local population density. This scenario is consequent with many existent-world autoinducers, especially those of Gram-negative bacteria and some unicellular yeasts, which are closely related to, or just are, metabolic waste products [4,fifteen,46,47]. Ane mode that the transition from a nonresponsive to a responsive quorum-sensing phenotype could occur is through mutation in a preexisting transcription factor, which allows information technology to bind the accumulating autoinducer. Binding the autoinducer may then alter the transcription gene's ability to control the expression of an EPS synthase. This abstraction conforms very well with the molecular mechanism underlying LuxI/R-type quorum-sensing circuits widely observed amidst bacteria [4,fifteen,48].

Bacteria grow according to Monod saturation kinetics, and nosotros assume that all cells secrete an autoinducer without price and at a abiding rate (Table 2). Following the pattern exhibited by V. cholerae, QS⁺ cells synthesize EPS only when local autoinducer concentration is below the quorum-sensing threshold concentration. Once this threshold level is exceeded, QS⁺ cells end EPS synthesis and invest just in biomass product [nine]. The timing and density dependence with which QS⁺ bacteria achieve a quorum depends upon three key factors: (1) how quickly the autoinducer is produced, (2) how quickly the autoinducer diffuses away from the biofilm, and (3) the critical quorum-sensing autoinducer concentration. For case, fast autoinducer product, slow autoinducer improvidence, and a depression critical quorum-sensing autoinducer concentration will all pb to a quorum being reached more speedily and at lower cell density. To account for the dependence of quorum-sensing behavior on all of these factors, we group them into a single parameter, , where σ is the autoinducer product charge per unit per unit bacterial biomass, D_AI is the autoinducer diffusion coefficient, and φ is the quorum-sensing threshold autoinducer concentration. ρ₁₀, the bacterial biomass density, and L, the length of the biofilm simulation infinite, are included in α to form a dimensionless group. Using a dimensionless group to describe the quorum-sensing process allows united states to brand qualitative predictions that are independent of the specific values of the parameters contained in α, albeit inside the bounds of systems that take these physical backdrop.

Strains with the aforementioned α value will reach their respective quorums at the same time afterward the initiation of biofilm growth, irrespective of the different potential combinations of σ, D_AI , φ, ρ_X, and 50 that can produce a particular α value. Although α accounts for multiple factors that simultaneously contribute to quorum-sensing dynamics, to aid intuition, one may hold all parameters other than φ abiding and think of α as the critical quorum-sensing autoinducer concentration. α simply measures how readily QS⁺ cells switch from low to high cell-density state: for higher α, QS⁺ bacteria will reach a quorum at higher cell density and relatively later on in the course of biofilm growth.

In order to determine whether simple quorum-sensing behavior (QS⁺) provides a fitness advantage over the unconditional behavioral strategies EPS⁺ and EPS⁻, we first consider competition in mixed biofilms initialized with the aforementioned number of either (1) QS⁺ and EPS⁺ or (2) QS⁺ and EPS⁻. We replicate these simulations over a range of α values for the QS⁺ strain in guild to examine how the timing and density dependence of quorum sensing influence the outcome of competition.

Simple Contest: QS⁺ versus EPS⁺, and QS⁺ versus EPS⁻

Simulations were parameterized with empirically estimated values (Table iii), initialized with fifty cells of each strain placed randomly on the solid substratum, and allowed to run for 14 simulated days (Figure one), which is close to the maximum elapsing of a Five. cholerae infection [49]. The proportion of energy invested in EPS secretion (f) will determine the extent to which EPS production allows i strain to displace others from a biofilm. As Xavier and Foster accept discussed [36], for a given set of simulation parameters, in that location exists some evolutionarily stable strategy for EPS product, f*, which volition out-compete any strain that invests either more or less in EPS. To find this optimum strategy, we performed an evolutionary stability analysis in which EPS⁺ strains with incrementally larger or smaller f values were competed against each other (see Text S1 and Figure S2). We constitute that, for our model atmospheric condition, the evolutionarily stable strategy for EPS investment independent of quorum sensing is approximately f* = 0.5, which was used for both the EPS⁺ and the QS⁺ strains (when below its quorum) in all subsequent simulations.

Figure 1. Direct Competition between QS⁺ and EPS⁺ Bacteria Initialized with Equal Numbers of Both Strains

Autoinducer (AI) concentration is shown in the groundwork, where isoconcentration lines represent 0.1-mg/fifty steps. Both strains behave identically, producing both EPS and biomass, until the autoinducer quorum-sensing threshold is reached. QS⁺ cells and so turn off polymer secretion, devote all resources to biomass production, and reach a growth burst at locations on the upper surface of the biofilm where substrate availability is highest. A moving picture for this simulation is provided as Video S1.

https://doi.org/10.1371/journal.pbio.0060014.g001

Competitions between the QS⁺ and EPS⁺ strains and between the QS⁺ and EPS⁻ strains were repeated for a range of α values. Nosotros included two controls, one (α = ∞) in which the QS⁺ strain never reaches its quorum and behaves identically to the EPS⁺ strain, and another (α = 0.001) in which the QS⁺ strain reaches a quorum immediately later simulations begin, and behaves identically to the EPS⁻ strain thereafter. The frequency of QS⁺ cells within the biofilm was calculated at each fourth dimension step and averaged over all replicate simulations to generate a mean QS⁺ frequency plot for each α value used in both sets of competitions (Figure 2A and 2B).

Effigy 2. Summary of Simple Competitions

(A) A quorum-sensing strain that downward-regulates polymer secretion at high jail cell-density (QS⁺) is competed against a constitutive polymer-secreting strain (EPS⁺).

(B) QS⁺ versus non-polymer producer (EPS⁻). Each competition (QS⁺ vs. EPS⁺, and QS⁺ vs. EPS⁻) was replicated 50 times for each of the α values: ∞, 0.01, 0.008, 0.005, and 0.001, where α captures how quickly the QS⁺ strain will switch from low to high cell-density state (meet main text). For higher α, QS⁺ bacteria volition reach their quorum at higher cell density, relatively later on during biofilm growth. Plotted lines represent mean QS⁺ frequency time series from each set of fifty simulations and are shown with shaded 95% confidence intervals. Annotation that in (A) and (B), the plotted lines corresponding to α = ∞ are control treatments in which QS⁺ behaves identically to EPS⁺ throughout simulations because autoinducer concentrations never reach the QS⁺ quorum-sensing threshold. Similarly, in (A) and (B), the plotted lines corresponding to α = 0.001 are command treatments in which QS⁺ behaves identically to EPS⁻ throughout simulations because autoinducer concentrations ever exceed the QS⁺ quorum-sensing threshold.

https://doi.org/10.1371/journal.pbio.0060014.g002

Competition betwixt QS⁺ and EPS⁺.

In a mixed competition between the quorum-sensing strain and a constitutive EPS producer, all cells are initially phenotypically identical; that is, they all secrete EPS. However, as cells grow and population density increases, the autoinducer accumulates, and at a time bespeak dependent upon their α value, quorum-sensing (QS⁺) cells plough off polymer secretion and invest all their resource in growth. Nearly the upper surface of the biofilm, where substrate availability is highest, QS⁺ cells achieve a burst of cell division (Figure 1, days 9–13). In the brusque term, the QS⁺ strain increases in frequency over and above that of the constitutive EPS producer. The advantage is temporary, nevertheless, because the EPS⁺ strain continues to secrete polymer and eventually produces towers that suffocate neighboring QS⁺ cells (meet for example Figure 2A, α = 0.005), analogous to the case of competing EPS⁺ and EPS⁻ cells [36]. Quorum-sensing command of EPS product, therefore, provides a competitive advantage over constitutive EPS production, but only for a limited time window. Moreover, the location of this window within the period of biofilm growth is determined past how quickly the QS⁺ strain reaches a quorum. Strains with higher α attain growth bursts later in the class of biofilm formation (Figure 2A).

Competition between QS⁺ and EPS⁻.

Without having to pay the cost of EPS production, EPS⁻ cells chop-chop divide at the first of simulations and achieve a higher initial frequency than QS⁺ cells. By secreting EPS, however, the QS⁺ strain rises up and over the top of neighboring cells, suffocating those that do not secrete polymer. After its initial disadvantage due to lower growth rate, the QS⁺ strain quickly ascends to a majority in the biofilm and remains in that location indefinitely. Unlike the EPS⁺ strain, QS⁺ cells switch to pure biomass production later they have suffocated their EPS⁻ neighbors; at this point, investment into EPS is no longer advantageous. Equally a issue, the QS⁺ strain volition out-compete non-EPS producers past even larger margins than the constitutive EPS producer (Figure 2B).

Rare-Mutant Invasion Assay

The unproblematic competition simulations described above propose that bacteria for which EPS production is under quorum-sensing control have a fourth dimension-dependent reward over strains that are not capable of responding to changes in population density. However, a within-grouping competitive advantage need non translate into evolutionary success when the reward comes at a strong cost to overall productivity [50]. More than concretely, if successfully suppressing another strain in a biofilm causes the entire biofilm to grow poorly, the cyberspace effect on fitness may be deleterious [36]. Nosotros investigated this possibility through evolutionary invasion analyses to determine whether rare-mutant QS⁺ cells tin increase in frequency in populations of either EPS⁺ or EPS⁻ cells, and whether a successful QS⁺ strain, once in the majority, tin can afterward resist invasion by rare EPS⁺ and EPS⁻ mutants. To do this, we simply compare the number of cell divisions of the invading strain in a focal biofilm to the mean number of cell divisions past the bulk strain taken across all biofilms in the population. More formally, we get-go define the fitness of a bacterial strain as the average number of cell divisions that it achieves on a divers fourth dimension interval [0, t_end ]: where N _{Due south,t} is the number of cells of strain S present within the biofilm at time t. Letting S1 be a rare mutant, we define its power to invade a majority strain, S2, equally follows: where w _S1 is the fitness of the potential invader (S1) in straight competition with S2, equally described in Equation ane, and is the mean fitness of S2 cells in a pure S2 biofilm, which approximates mean fitness in the population. We assume that the bacterial population as a whole contains many more biofilms than the focal faux biofilm in which the potential invading strain (S1) has arisen. All biofilms other than the focal simulated biofilm are composed purely of the resident strain, S2, and contribute vastly more than to hateful population fitness. Therefore, effectively measures the fettle of S2 cells when competing solely with other S2 cells. For a rare-mutant S1 to invade a majority strain S2, must exist greater than unity; that is, S1 must fare ameliorate against S2 than S2 fares against itself [51].

Length of biofilm tenure: A primal variable in this assay is the time interval [0, t_terminate ] on which due west _S1 and are measured. When choosing t_end , we are asking: at what point during biofilm growth is it critical for long-term evolutionary success to be in the majority? Nosotros take the answer to be the fourth dimension at which dispersal or disturbance occurs, and we presume that all cells within a biofilm take an equal probability of entering the propagule pool from which subsequent biofilms are seeded. This arroyo takes into consideration both local competition within biofilms and global contest betwixt biofilms to determine the long-term evolutionary success of an invading bacterial strain [l]. Chiefly, our method of analyzing invasiveness also assumes that dispersal or disturbance occurs in 1 large outburst at a discrete point in time, rather than continuously throughout biofilm growth (see Give-and-take).

Genetic relatedness at biofilm initiation: We performed reciprocal invasion analyses using simulated competitions between QS⁺ and EPS⁺ or QS⁺ and EPS⁻ with a range of initial QS⁺ frequencies. This captures the issue of a rare mutant inbound a population of some other strategy, where the starting frequency of the rare strain reflects the number of strains randomly inoculated, and therefore the initial average relatedness, within the biofilm. For example, if ten strains are nowadays at the initiation of each biofilm, and then a rare mutant will begin at a local frequency of 0.1, and average relatedness inside the biofilm where the rare mutant resides will outset at 0.1 [2,36].

Invasion analysis: QS⁺ and EPS⁺.

We investigated whether a quorum-sensing strain that obtains an advantage in unmarried biofilms (Figures 1 and 2) tin invade a population of constitutive EPS producers and resist their reinvasion. We therefore focus on parameter values nether which the QS⁺ strain has an advantage in the uncomplicated competition simulations. Specifically, we examine invasiveness for a disturbance interval of 9 d (t_end = 9), with a QS⁺ strain α value (QS sensitivity) of 0.008, and we find that the QS⁺ strain can readily invade populations composed mostly of EPS⁺ cells, only not vice versa (Effigy 3A and 3B). Additionally, biofilms composed entirely of QS⁺ cells have a college average fitness than biofilms composed entirely of EPS⁺ cells.

Effigy 3. The Quorum-Sensing Strain Can Invade Non-Quorum-Sensing Strains, only Not Vice Versa

Invasiveness of a rare mutant was analyzed for dissimilar degrees of mixing among strains in biofilms, reflected in the different initial frequencies of the rare strain in the biofilm. For example, if 10 strains are randomly sampled, and then the initial frequency of the rare mutant in its own biofilm will be 0.1; initial relatedness will besides be 0.1 (encounter principal text). Each box and whisker plot summarizes the results of 20 replicate simulations, and plus signs (+) announce outliers. All simulations were carried out at α = 0.008 for the QS⁺ strain.

(A) Invasion of a rare quorum-sensing strain (QS⁺) into a population of unconditional EPS producers (EPS⁺), and (B) failure of a rare EPS⁺ strain to invade a population of QS⁺ bacteria. Biofilms composed entirely of QS⁺ cells accomplish college average fitness than biofilms composed entirely of EPS⁺ cells.

(C) Invasion of a rare QS⁺ strain into a population of non-EPS producers (EPS⁻), and (D) failure of a rare EPS⁻ strain to invade a population of QS⁺ bacteria. Again, the QS⁺ strain can invade EPS⁻, whereas EPS⁻ cannot invade QS⁺. Notably, however, biofilms equanimous entirely of QS⁺ cells have a lower average fitness than biofilms equanimous entirely of EPS⁻ cells. Therefore, if all biofilms contained merely a single genotype (no within-biofilm evolutionary contest), the EPS⁻ would invade and resist invasion.

https://doi.org/10.1371/journal.pbio.0060014.g003

Invasion analysis: QS⁺ and EPS.⁻.

Again using t_finish = 9 d, we observe that the QS⁺ strain invades a resident population of EPS⁻ cells, whereas the reverse is not true (Figure 3C and 3D). It is notable, however, that biofilms composed entirely of QS⁺ cells have a lower mean fitness than biofilms equanimous entirely of EPS⁻ cells, which reflects the fact that investment into EPS reduces total biomass production and therefore average growth rate.

Discussion

Biofilm formation and quorum sensing are central and often interconnected features of bacterial social life [4–half-dozen,xv,28,29]. Our evolutionary analysis is the first to address both of these major classes of bacterial social beliefs, and it suggests that quorum sensing enables leaner to plough on and off the secretion of extracellular polymeric substances (EPS) so as to increase their competitive ability confronting other species and strains within biofilms. This result builds upon the conclusions of Xavier and Foster [36], who predicted that EPS secretion affords an advantage to secreting strains in competition with nonsecreting strains. Information technology is important to note that this view contrasts with the conventional wisdom that EPS is a public good that only binds the biofilm together and protects it confronting external threats [52,53], although a combination of these perspectives is besides a realistic possibility. We modeled both positive and negative quorum-sensing regulation of EPS product. Though explicit simulations of the planktonic phase were omitted, it seems articulate that secreting EPS at loftier cell density allows cells to selectively actuate EPS synthesis in biofilms and avoid the price of EPS production in the planktonic phase [36]. Notwithstanding, nosotros also observe potential benefits for down-regulating EPS at high cell density, which allows cells to redirect free energy from EPS production into growth and cell division prior to a dispersal upshot (Effigy ane). Such a quorum-sensing phenotype will merely exist favored if disengagement events are predictable, due to consistent extrinsic disturbance, or if dispersal is induced by the leaner themselves.

Our findings are consistent with the known biology of V. cholerae, which exhibits negatively quorum-sensing–regulated EPS secretion. The environments that 5. cholerae occupies appear to present opportunities for EPS-mediated competition within biofilms. Although we do not all the same know how oftentimes different Five. cholerae strains compete within human hosts, it is clear that infections always involve multiple species. These include other pathogenic genera, such as Pseudomonas, Salmonella, and Campylobacter [54,55], and there is compelling evidence that Five. cholerae must compete with native intestinal microbial fauna in order to become established [56]. Furthermore, EPS secretion appears to be important for within-biofilm contest: quorum-sensing–scarce V. cholerae mutants that overproduce EPS take over biofilm cultures coinoculated with wild-type bacteria [9].

The environmental of pathogenic V. cholerae is characterized past cycles of rapid growth followed by massive dispersal events; the bacteria effect a stereotypical disease progression from initial infection, through the formation of biofilm-like aggregates [57], to release from the intestinal tract afterward enormous toxin-induced fluid release. This suggests that quorum sensing in V. cholera can be tuned to coincide with purging from the gut. Interestingly, quorum-sensing mutants that overproduce EPS suffer a greatly decreased ability to escape from biofilms [58–60], which indicates that, in improver to saving energy, reducing EPS secretion also actively assists dispersal. Moreover, on reaching a quorum, V. cholerae produces a protease whose putative function is to facilitate detachment [9,39,58,59]. By secreting a "detachase" and down-regulating EPS production at loftier cell density, V. cholerae appears to exist inducing a growth outburst coincident with efficient dispersal. The cycle of growth and detachment may likewise play a role in the initial colonization of the host: cells in a biofilm formed early in an infection tin can, upon detecting a threshold autoinducer concentration, halt EPS secretion, detach, and seed other areas of the intestine.

Whereas V. cholerae terminates EPS secretion at high cell density, many other species, including the opportunistic human pathogen P. aeruginosa, activate EPS secretion at high prison cell density. Hammer and Bassler [ix] suggested that the explanation for this stark dissimilarity in quorum-sensing behavior may lie in dissimilar infection strategies. Our results back up this argument and, furthermore, suggest that this divergence hinges upon the evolutionary tradeoff between inside-biofilm contest on the one hand and dispersal ability on the other. In particular, chronic infections are less likely to involve detached and anticipated moments of detachment that would favor a articulate cutoff point for polymer secretion. Instead, dispersal is likely to occur through many small events over a long, but indeterminate, period of time. In such weather condition, strains that can dominate locally, thereby maximizing their chances of disengagement over an interval of uncertain length, will accomplish an evolutionary advantage. We therefore predict that up-regulation of EPS secretion at loftier cell density, which focuses resources investment into sustained local competitive ability, is more likely to be favored. This is precisely the pattern exhibited by P. aeruginosa, which is notorious for the chronic, and often terminal, infections it establishes in the lungs of cystic fibrosis patients. Interestingly, populations of P. aeruginosa sampled from the cystic fibrosis lung ofttimes besides incorporate quorum-sensing mutants that are fixed in a loftier cell-density land [61] and a low cell-density land [33,62], although the link between these results and the EPS secretion phenotype, if whatsoever, is non yet articulate.

The biofilm simulations performed in this study highlight several hypotheses amenable to experimental testing. We anticipate that EPS production past Five. cholerae is at to the lowest degree partially a competitive beliefs in the human intestinal tract, as it is in lab biofilm assays [ix]. Although nosotros lack a direct test of this prediction, Nielsen et al. [sixty] institute that V. cholerae mutants unable to produce EPS are just every bit effective at colonizing rabbit intestine equally wild-type cells, which shows that EPS is not secreted just to aid surface colonization. The same study establish that rpoS, which encodes an important stationary-phase regulator, is necessary for escape from the intestinal wall, implying that the detection of nutrient starvation too regulates dispersal [60].

A comparing of different 5. cholerae strains offers additional opportunities to test our conclusions. Natural isolates bear witness considerable variation in quorum-sensing power, with strains fixed in either low or high cell-density states [63]. Our simulations raise the possibility that variation in quorum-sensing land within Five. cholerae is linked to different dispersal requirements across the bacterium'southward diverse ecology. Five. cholerae strains are known to form biofilms on both biotic and abiotic surfaces in marine environments [64–66], and not all crusade disease [63]. Specifically, we predict that pathogenic strains selected for rapid colonization of, and efficient dispersal from, human hosts or other temporary environments will exhibit negatively quorum-sensing–regulated EPS production. The Classical Five. cholerae biotype, which was responsible for the get-go six global cholera pandemics, has a nonfunctional re-create of a cardinal regulatory poly peptide, HapR, involved in the quorum-sensing response. However, in line with our predictions, it was recently discovered that these strains are capable of HapR-contained quorum sensing and may notwithstanding repress EPS expression in response to high cell density [67]. The associated prediction is that strains that occupy unmarried locations for long periods should accumulate mutations that enable constitutive EPS product in biofilms, regardless of local population density. In support of this, standing cultures of EPS⁻ V. cholerae cells are reliably taken over by spontaneous, constitutive EPS⁺ mutants [9].

Cooperation, contest, and communication are all intertwined in microbial communities, and we are only start to unravel the processes that drive this rich interaction [1,2,68,69]. Although our simulations inevitably miss many biological details of whatsoever one species or strain, a familiar principle of sociobiology emerges. A full agreement of quorum sensing in bacterial biofilms will crave consideration of evolutionary competition within and among these social groups.

Supporting Information

Effigy S1. Summary of Simple Competition Involving the QS* Strain, Which Up-Regulates Polymer Secretion at High Density

(A) Competition between the QS* strain and the constitutive EPS-secreting strain (EPS⁺).

(B) Competition between the QS* strain and the non–EPS-secreting strain (EPS⁻). These simulations differ from those carried out for Figure 2 (master text); hither, the QS* strain produces no EPS at low cell density and initiates EPS secretion only afterward autoinducer concentration exceeds the threshold value. Each competition was repeated 50 times, and plotted lines represent mean QS* frequency fourth dimension series from each set up of simulations, shown with shaded 95% confidence intervals.

ten.1371/journal.pbio.0060014.sg001

(1.0 MB EPS)

Figure S2. An Evolutionary Stability Assay for Investment into EPS (f)

Each box-and-whisker plot summarizes the results of 20 replicate simulations.

(A) Invasion analysis (see Equation 1, main text) of EPS⁺ strains with slightly college f values than the balance of the population (f − Δf) yields f* = 0.52.

(B) Invasion analysis of EPS⁺ strains with slightly lower f values than the rest of the population (f + Δf) yields f* = 0.45. Together, these 2 analyses demonstrate that the evolutionarily stable strategy for EPS investment, f*, lies between 0.45 and 0.52, and f = 0.5 was used for the simulations in our primary text. The value of Δf used for this evolutionary stability analysis was 0.i. Focal biofilms were initiated with an equal number of cells of each type (boilerplate relatedness of 0.5), and invasiveness was calculated using t_terminate = fourteen d (see main text).

x.1371/journal.pbio.0060014.sg002

(502 KB EPS)

Text S1. Simulation of a Bacterial Strain that Up-Regulates EPS Production (QS*) at High Cell Density in Competition with Constitutive EPS Producers (EPS⁺) and Non-Producers (EPS⁻), and an Evolutionary Stability Analysis for Investment into EPS Secretion

10.1371/periodical.pbio.0060014.sd001

(44 KB Doctor)

Acknowledgments

We are very grateful to Bonnie Bassler, Katharina Ribbeck, Ned Wingreen, Karina Xavier, Adrian de Froment, Jonathan Dushoff, Andy Gardner, and two anonymous reviewers for comments on this manuscript. Nosotros as well thank Brian Hammer for invaluable discussions that helped to motivate this project, and Iain Couzin for organizing the meeting that led to our collaboration.

Author Contributions

CDN, JBX, and KRF designed simulations. CDN and JBX performed simulations. CDN, JBX, and KRF analyzed information. JBX contributed belittling tools. CDN, JBX, SAL, and KRF wrote the newspaper.

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